CBS上调P2X3受体表达参与腰椎间盘突出症模型大鼠持续性痛觉高敏

发布时间：2018-05-25 19:44

本文选题：腰椎间盘突出症 + 神经病理性疼痛　；参考：《苏州大学》2015年硕士论文

【摘要】：目的1.探究P2X3受体在大鼠腰椎间盘突出诱导的神经病理性疼痛中的作用;2.探讨H2S信号分子在调节L5/L6背根神经节P2X3受体表达和功能中的作用特征。方法1.采用200-220g SD雄性大鼠,取出自体尾椎的正常髓核组织,置于手术显露后的腰5,6左侧神经根处,建立腰椎间盘突出症动物模型(LDH)。分别在术前1天,术后3,7,14,21,28和35天运用VFF(Von Frey filaments)及双足平衡实验(Weight Bearing)的方法来评测SD大鼠的行为学疼痛阈值反应。2.运用逆行标记的荧光素(Dil)来标记支配后肢的L5/L6背根神经节(DRG)中、小神经元,运用钙成像方法研究腰椎间盘突出症(LDH)大鼠L5/L6 DRG神经元中ATP诱导的细胞内钙信号反应及神经元的兴奋性变化。3.运用Western Blotting方法检测LDH模型大鼠L5/L6 DRG神经元中内源性硫化氢合酶CBS和CSE以及嘌呤受体P2XRs的蛋白表达情况。4.运用免疫组织化学方法检测LDH大鼠L5/L6 DRG神经元中CBS和P2X3R蛋白共表达情况。结果1.自体尾椎髓核(NP)移植能够显著降低LDH模型大鼠后肢的机械性疼痛阈值,并且这一现象是与L5/L6 DRG神经元中硫化氢合酶CBS蛋白的表达上调相关联的。2.与假手术组(Sham)相比,LDH组增加了L5/L6背根神经节(DRG)中P2X3R的蛋白表达量,而P2X2R,P2X1R的蛋白表达量并没有发生变化。3.对LDH大鼠鞘内注射硫化氢合酶CBS抑制剂AOAA后,发现AOAA能够显著的翻转LDH大鼠的疼痛阈值,并呈现剂量和时间上的依赖性。而作为对照,AOAA并不能够对假手术组SD大鼠的疼痛阈值产生作用。4.对LDH大鼠鞘内注射P2X3R的选择性拮抗剂A-317491后,发现A-317491能够显著的翻转LDH大鼠的疼痛阈值,并呈现剂量和时间上的依赖性。5.运用免疫组化的实验方法检测后发现硫化氢合酶CBS与P2X3R在L5/L6神经元中能够共表达。而且运用Western Blotting的方法进行检测后发现,连续给予LDH大鼠AOAA能够显著翻转NP引起的L5/L6神经元中的P2X3R表达的上调。6.全细胞膜片钳记录显示,与Sham组相比,在LDH大鼠L5/L6 DRG上,NP能显著增强ATP诱导的细胞内钙信号。鞘内连续给与硫化氢合酶CBS抑制剂AOAA后可以抑制LDH大鼠中ATP诱导的钙信号变化。7.通过单次或连续给与LDH大鼠足趾注射硫化氢合酶CBS抑制剂AOAA后,发现AOAA不能够翻转LDH大鼠的疼痛阈值,而且运用Western Blotting的方法进行检测后发现,连续足趾给药后大鼠L5/L6神经元中P2X3R表达未发生变化。结论腰椎间盘突出症模型(LDH)中髓核(NP)激活了内源性硫化氢(H2S)合酶CBS表达的上调,通过大鼠DRG中初级感觉神经元,调节了L5/L6神经元中P2X3受体蛋白的表达,从而增加L5/L6神经元的兴奋性进而产生神经病理性疼痛;我们的研究结果表明,内源性硫化氢(H2S)合酶CBS表达的上调可能在LDH大鼠椎间盘源性疼痛过程中扮演着重要的角色。因此,本研究能够在一定程度上揭示根源性疼痛产生的分子机制,从而为神经病理性疼痛的治疗提供更为有效的治疗方案。
[Abstract]:Objective 1. To explore the role of P2X3 receptor in neuropathic pain induced by lumbar disc herniation in rats. To investigate the role of H _ 2S signaling molecules in regulating the expression and function of P2X3 receptor in L5/L6 dorsal root ganglion. Method 1. The normal nucleus pulposus of the autologous tail vertebrae was removed from 200-220g SD male rats and placed at the left nerve root of the left lumbar vertebrae after operation. The animal model of lumbar disc herniation was established. The behavioral pain threshold responses of SD rats were evaluated by VFF(Von Frey filaments and weight balance test 1 day before operation, 3 days after operation, and 35 days after operation. Small neurons in the dorsal root ganglion of L5/L6, which innervate the hind limbs, were labeled with retrograde fluorescein Dil. Calcium imaging was used to study the intracellular calcium signal response induced by ATP and the changes of neuronal excitability in L5/L6 DRG neurons of rats with lumbar disc herniation. The expression of endogenous hydrogen sulfide synthase (CBS) CSE and purine receptor P2XRs (P2XRs) in L5/L6 DRG neurons of LDH model rats was detected by Western Blotting method. The co-expression of CBS and P2X3R in L5/L6 DRG neurons of LDH rats was detected by immunohistochemical method. Result 1. Autologous transplantation of nucleus pulposus of caudal vertebrae can significantly reduce the threshold of mechanical pain in the hind limbs of LDH model rats, and this phenomenon is associated with the up-regulation of CBS protein expression in L5/L6 DRG neurons. Compared with sham group, the expression of P2X3R protein was increased in L5/L6 dorsal root ganglion (DRG), but the protein expression of P2X2RnP P2X1R did not change. After intrathecal injection of AOAA, a CBS inhibitor of hydrogen sulfide synthase, in LDH rats, it was found that AOAA could significantly reverse the pain threshold of LDH rats in a dose-and time-dependent manner. AOAA, as a control group, had no effect on the pain threshold of SD rats in sham-operated group. After intrathecal injection of P2X3R selective antagonist A-317491 to LDH rats, it was found that A-317491 could significantly reverse the pain threshold of LDH rats, and showed a dose-and time-dependent dependence. It was found that CBS and P2X3R co-expressed in L5/L6 neurons by immunohistochemistry. Moreover, by using Western Blotting method, it was found that the up-regulation of P2X3R expression in NP-induced L5/L6 neurons could be significantly reversed by continuous administration of AOAA in LDH rats, and the up-regulation of P2X3R expression in NP-induced L5/L6 neurons was significantly reversed. Whole cell patch clamp recording showed that NP on L5/L6 DRG of LDH rats could significantly enhance intracellular calcium signal induced by ATP compared with Sham group. Intrathecal administration of AOAA, a CBS inhibitor of hydrogen sulfide synthase, inhibited the changes of calcium signal induced by ATP in LDH rats. After a single or continuous injection of AOAA, a CBS inhibitor of hydrogen sulfide synthase, into the toes of LDH rats, it was found that AOAA could not reverse the pain threshold of LDH rats, and that AOAA could not reverse the pain threshold of LDH rats. There was no change in P2X3R expression in L5/L6 neurons of rats after continuous toe administration. Conclusion NPN in nucleus pulposus of lumbar disc herniation model can activate the up-regulation of CBS expression of endogenous hydrogen sulfide (H2S) synthase, and regulate the expression of P2X3 receptor protein in L5/L6 neurons through primary sensory neurons in rat DRG. Our results suggest that the up-regulation of CBS expression of endogenous hydrogen sulfide (H2S) synthase may play an important role in the process of discogenic pain in LDH rats. Therefore, to some extent, this study can reveal the molecular mechanism of root pain, thus providing a more effective treatment for neuropathic pain.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R681.5

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